Enhancing the anti-tumor effect of melphalan with L-amino acid oxidase

ABSTRACT

L-amino acid oxidase is utilized to reduce plasma level of large neutral amino acids to allow the opportunity of increased melphalan transport into tumors and melphalan is administered when the plasma level of L-amino acid oxidase is sufficiently low so the gain from increased transport outweighs the loss from L-amino acid oxidase-mediated metabolism of melphalan.

This invention was made at least in part with Government support underNational Institutes of Health grant number DK 26912. The Government hascertain rights in the invention.

TECHNICAL FIELD

This invention is directed at the use of melphalan as an anti-tumoragent.

BACKGROUND OF THE INVENTION

Melphalan, (4-[bis(2-chloroethyl)amino]-L-phenylalanine), is a nitrogenmustard that is useful as a chemotherapeutic agent against many tumors.With cells grown in culture, it is effective against many brain tumors.However, it is poorly transported across the blood brain barrier andthus has not been found effective for use against many brain(intracranial) tumors.

It has been discovered that starving followed by a protein-free dietreduces plasma levels of protein amino acids including large neutralamino acids and increases the blood-to-tumor periphery tissue transferconstant of melphalan both for subcutaneous tumors (representative ofall tumors except for brain tumors) and also for brain tumors. SeeFriedman, H. S., et al, Proceedings of the American Association forCancer Research, Volume 32, page 318, Abstract 1886, March, 1991. Thisincrease of blood-to-tumor tissue transfer constant might be expected toallow use of lesser dosages of melphalan (and concomitant reducedtoxicity) in circumstances where melphalan is now considered useful andthe extension of use of melphalan in circumstances now foreclosed by theblood brain barrier, i.e., as an anti-tumor agent against brain(intracranial) tumors. However, the accomplishment of this by means ofstarving and administration of a protein free diet affords at mostlimited improvement.

The finding that reduced plasma levels of large neutral amino acids wereassociated with increased blood-to-tumor melphalan transfer constants isconsistent with previous work showing that melphalan is transported bythe same transporter as the large neutral amino acids and that thepresence of large neutral amino acids in plasma interferes with thetransport of melphalan. Thus achieving reduction of plasma levels oflarge neutral amino acids by means different from or additional torestricted diet to the same or greater degree as is obtained with saidrestricted diet, should improve melphalan transport and result in abenefit if said different means doesn't concurrently provide deleteriouseffect.

Various enzymes are known for which large neutral protein amino acidsare substrates and which would be useful for reducing plasma levels ofthese provided they have access to required cosubstrates. However,melphalan is also an amino acid and would likely be a substrate for thesame enzymes. Furthermore, the various possible enzymes would beexpected to differ in respect to the number of different large neutralamino acids that would be substrates and in their relative kineticconstants vis-a-vis their large neutral amino acid substrates andmelphalan. Therefore, such enzymes might be expected, on the one hand,to potentiate the transport of melphalan into tumors by reducingconcentrations of plasma large neutral amino acids but on the otherhand, would be expected to act in counterproductive fashion by degradingmelphalan to an extent which might be larger than the extent ofincreased melphalan transport from large neural amino acid depletion.Furthermore, each particular enzyme might be expected to have differenteffects on concentrations of plasma large neutral amino acids and thedegradation of melphalan. What is necessary is selection of an enzymewhich will reduce plasma large neutral amino acids to enhance melphalantransport but which would be relatively less active toward melphalan orwhich would be sufficiently inactivated, within the period of reducedplasma amino acid level, so as not to degrade the melphalan to an extentof negating the benefit obtained by enhanced melphalan transport. Forany particular enzyme, there is no expectation of success of meetingthis criterion.

SUMMARY OF THE INVENTION

It has been discovered herein that L-amino acid oxidase meets thiscriterion and successfully enhances the delivery of melphalan to obtainimproved effect at conventional melphalan dosages in treatment ofsubcutaneous tumors, and to provide enhanced transport of melphalanacross the blood brain barrier to obtain improved anti-tumor effectagainst brain tumors, and when accompanied by fasting and/or a proteinrestricted diet provides improved efficacy over fasting and proteinrestricted diet alone. In contrast to starving which reduces the plasmalevel of essentially all amino acids, use of L-amino acid oxidasereduces the plasma level of only amino acids that are actively degradedby L-amino acid oxidase, namely, plasma large neutral amino acids asdefined hereinafter and provides α-ketoacid reaction products which areeventually converted back to amino acids in the body or are catabolizedintracellularly for energy.

It has been discovered herein that improved anti-tumor effect isobtained by utilizing L-amino acid oxidase to reduce plasma level oflarge neutral protein amino acids to allow the opportunity of increasedmelphalan transport into tumors and then capitalizing on thisopportunity by administering melphalan when the plasma level of L-aminoacid oxidase activity is sufficiently low so the gain from increasedtransport outweighs the loss from L-amino acid oxidase-mediatedmetabolism of melphalan.

The method herein is directed to treating a subject (human patient oranimal) for tumors and comprises the steps of

(a) administering L-amino acid oxidase to said subject at a dosageranging from about 1 to about 100 units/ml of plasma (corresponding toabout 85 to about 8500 units/kg of body weight), which is non-toxic andwhich is sufficient to reduce plasma level of large neutral amino acidsfrom a normal level to a melphalan transport improving level;

(b) administering melphalan in an anti-tumor effective amount to thesubject wherein plasma level of large neutral amino acids remainsreduced from normal level within about 2 to 36 hours after administeringin step (a), so as to improve the transport of the melphalan into thetumors, as evidenced by increase in number of subjects surviving pastcontrol or increased median survival time of subjects compared to whenmelphalan is administered without L-amino acid oxidase beingadministered first.

In a preferred method, the L-amino acid oxidase is administered in step(a) at a dosage ranging from about 10 units/ml to about 50 units/ml andthe administration of step (b) is carried out from 12 to 30 hours afterthe administration of step (a).

In a very preferred embodiment, the method herein comprises preventingreplenishment of large neutral amino acids to plasma during the periodbetween the administration of step (a) and the administration of step(b) and optimally also during the period of melphalan uptake into tumorsby causing the patient to fast or by administering a protein-free dietor by causing the patient to fast followed by administering aprotein-free diet.

One unit of L-amino acid oxidase is that amount which converts 1 μmol ofL-leucine to α-ketoacid reaction product per minute at 37° C. andcorresponds approximately to 0.01 mg of crystalline L-amino acidoxidase.

The term "large neutral amino acids" is used herein to mean the group ofamino acids consisting of leucine, methionine, phenylalanine, tryptophanand tyrosine.

The term "normal level" of large neutral amino acids is used herein tomean the concentration of each of the large neutral amino acids found ina subject (patient or animal) not experiencing dietary restriction orsupplementation.

The term "melphalan transport improving level" is used herein to mean aconcentration of the large neutral amino acids in a subject (patient oranimal) that allows melphalan transport into the tumor to be improvedrelative to the extent of melphalan transport into the tumor that wouldbe observed in the absence of L-amino acid oxidase and large neutralamino acid depletion.

Amino acids can be qualified using standard techniques on a model 7300amino acid analyzer (Beckman Instruments, Inc., Palo Alto, Calif.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts graphs of L-amino acid oxidase plasma concentration(denoted "Active LOX Plasma Concentration") vs. time after intravenoustreatment of mice with 200 and 300 μg of L-amino acid oxidase, based ondata determined in Example II.

FIG. 2 depicts graphs of L-amino acid oxidase plasma concentration(denoted "LOX plasma concentration") vs. time after intravenoustreatment of mice with various doses of L-amino acid oxidase, showingplasma L-amino oxidase activity at denoted intervals after acid oxidasetreatment, based on data determined in Example III.

FIG. 3 depicts graphs of fraction of total radioactivity versus time,based on data determined in Example IV directed to HPLC determination ofmelphalan (50 μM) reaction with L-amino acid oxidase (0.25 mg/ml) and acontrol with no L-amino acid oxidase.

DETAILED DESCRIPTION OF THE INVENTION

L-amino acid oxidase is a flavoprotein that catalyzes the oxidativedeamination of certain L-amino acids to the corresponding α-keto acids.It occurs in many snake venoms (e.g., the venom of the EasternDiamondback rattlesnake) and is isolated therefrom as described inWellner, D., et al, J. Biol. Chem. 235,2013 (1960). For its use in themethod herein, the L-amino acid oxidase should be pure, i.e., free ofall of the toxins and other enzymes present in the snake venom fromwhich it is isolated. The required purification is accomplished, forexample, by isolating the compound in crystallized form by dissolving 1gm lyophilized snake venom (Miami Serpentarium) in 100 ml water, adding10 ml of 100 mM L-leucine, heating under N₂ to 70° C. for 5 minutes,centrifuging to remove precipitated protein, mixing the supernatantsolution with 400 mg (dry weight) of hydroxyapatite gel, adding HCl toreduce the pH to 5.5, centrifuging to obtain a precipitate of enzymebound to gel, resuspending the precipitate in 35 ml of 2.3M ammoniumsulfate in 80 mM Na acetate buffer, pH 4.6, to release the enzyme fromthe gel, centrifuging to obtain a supernatant containing enzyme, adding141 mg/ml of ammonium sulfate to precipitate the enzyme, centrifuging toobtain a pellet of the precipitate, dissolving the pellet in cold water,dialyzing the resulting solution against water at 4° C. to causecrystallization of the L-amino acid oxidase, collecting the crystals bycentrifugation, redissolving in 2 ml 100 mM KCl, again dialyzing againstwater to form crystals and then recrystallizing again. The crystals canbe dissolved in physiological saline to give an injectable preparation.

As indicated above, the L-amino acid oxidase is administered at a dosageranging from about 1 to about 100 units/ml of plasma (corresponding toabout 85 to about 8500 units/kg of body weight) which is non-toxic andwhich is sufficient to reduce plasma level of large neutral amino acidsfrom a normal level to a melphalan transport improving level. The dosagefor the L-amino acid oxidase can range, for example, from about 5 toabout 25 units/ml of plasma (corresponding to about 425 to about 2125units/kg of body weight) to reduce plasma level of large neutral aminoacids in a patient to a melphalan transport improving level of less than50% of normal level, and from about 10 to about 50 units/ml of plasma(corresponding to about 850 to about 4250 units/kg of body weight) toreduce plasma level of large neutral amino acids in a patient to amelphalan transport improving level of less than 10% of normal level.

The L-amino acid oxidase can be administered by any parenteral route,e.g., intravenously, intraperitoneally or subcutaneously. A preferredmethod of administration is intravenous administration.

The L-amino acid oxidase can also be administered to the patient byattaching it to an extracorporeal reactor and passing the patient'sblood through the reactor. Said reactor can be a packed bed of Dacronfibers to which L-amino acid oxidase is attached usingγ-aminopropyltriethoxysilane and glutaraldehyde (general method of R. Y.C. Ko, et al, J. Biomed. Res., 10, 249-258(1976)) or said reactor may bean insoluble carrier matrix of reconstituted bovine collagen containingL-amino acid oxidase (general method of L. S. Olanoff, et al, J. Biomed.Res., 8, 125-136(1977)) or said reactor may be a conventionalhollow-fiber hemodialyzer to which L-amino acid oxidase is attachedcovalently (general method of J. A. Jackson, et al, J. Pharmacol. Expt.Ther., 209, 271-274(1979)). In each case the patient's blood is passedthrough the extracorporeal reactor by means of conventionalarteriovenous cannulation wherein blood is removed from the patientthrough an arterial cannula, passed through the extracorporeal reactorand then returned to the patient through a venous cannula. Use of anextracorporeal reactor as described above, and disconnecting it prior tomelphalan administration, eliminates the opportunity for L-amino acidoxidase to degrade melphalan.

The L-amino acid oxidase is also advantageously administered inmethoxypropylene glycol (PEG) modified form, to increase its plasmahalf-life and to decrease its antigenicity. The PEG utilized ispreferably of 5000 daltons. The L-amino acid oxidase and PEG arecovalently coupled using 2,4,6-trichloro-s-triazine (general method ofK. V. Savoca, et al, Biochem. Biophys. Acta, 578, 47-53(1979)) and PEGis attached to 50% to 60% of the free amino groups of L-amino acidoxidase. L-amino acid oxidase modified in this manner has a circulatinghalf-life more than 10-fold that of unmodified L-amino acid oxidase. Theterm "utilizing L-amino acid oxidase" used herein includes usingunmodified L-amino acid oxidase as well as using PEG-modified L-aminoacid oxidase.

Turning now to step (b), as indicated above the dosage of melphalanadministered is an anti-tumor effective amount. Normally, this rangesfrom about 2 to about 120 mg/kg of body weight (corresponding to about 6to about 360 mg/square meter of body surface). Preferably, this rangesfrom about 5 to about 70 mg/kg of body weight (corresponding to about 15to about 210 mg/square meter of body surface), very preferably fromabout 11.8 to about 23.7 mg/kg (corresponding to about 35.5 to about 71mg/square meter of body surface).

As indicated above, administration in step (b) is carried out when theplasma level of large neutral amino acids remains reduced from normallevel so as to improve the transport of the melphalan into the tumors.When L-amino acid oxidase is administered parenterally in unmodifiedform, the administration of step (b) is ordinarily carried out from 2 to36 hours, very preferably from 12 to 30 hours, after the administrationof step (a). When the embodiment is used where L-amino acid oxidase islinked to an extracorporeal reactor, L-amino acid oxidase mediateddegradation of melphalan is not a factor and therefore time need not beprovided for L-amino acid oxidase inactivation to a level where it willnot degrade melphalan to too great an extent; in this case melphalan ispreferably administered at the earlier times in the above range. Whenthe embodiment is used where the L-amino acid oxidase is administered inPEG-modified form, the period for melphalan administration can beshifted to accommodate for the increased effective life of the L-aminoacid oxidase, e.g., to range from 12 to 360 hours after administrationof L-amino acid oxidase, with melphalan given, in some cases, more thanonce during that interval.

Preferably the melphalan is administered in an anti-tumor effectiveamount to the patient wherein the plasma large neutral amino acids areat the reduced level, when the patient's plasma L-amino oxidase level issuch that less than 15% of the melphalan would be metabolized by theL-amino acid oxidase during the period in which melphalan uptake intothe tumors is at least 85% complete. Very preferably, the melphalan isadministered when the patient's plasma L-amino acid oxidase level issuch that less than 10% of the melphalan would be metabolized by theL-amino acid oxidase during the period in which melphalan uptake intothe tumors is at least 90% complete. The period during which thepatient's plasma level is such that metabolizing of melphalan isrestricted to the recited degree and melphalan uptake into tumors iscomplete to the recited degree, may be estimated from data on L-aminoacid oxidase concentration in plasma versus time after administration,data on the rate of melphalan degradation at any L-amino acid oxidaseplasma concentration coupled with the assumption that L-amino acidoxidase mediated melphalan degradation is expected to be directlyproportional to the L-amino acid oxidase concentration and data knownfrom clinical studies on the time required for uptake of melphalan intovarious kinds of tumors to the specified amount of completeness modifiedby the estimate of reduction of the time as a result of the L-amino acidoxidase treatment.

Conventionally melphalan is administered orally. This is a suitablemethod of administration herein and is a preferred method ofadministration when the embodiment relying on PEG-modified L-amino acidoxidase is utilized. However, when L-amino acid oxidase in unmodifiedform is administered parenterally or is administered via anextracorporeal reactor, the melphalan is preferably administeredparenterally, very preferably intravenously as a bolus injection.

We turn now to the embodiment where replenishment of large neutral aminoacids to plasma is prevented during the period between theadministration of step (a) and the administration of step (b). This canbe carried out, for example, by precluding the patient from intake ofnutriments providing large neutral amino acids to the plasma essentiallyall during the period between the administering of step (a) and theadministering of step (b), e.g., by causing the patient to fast or byadministering a protein-free or protein-restricted diet during saidperiod. Suitable protein-free or protein-restricted diets include, forexample, standard total parenteral nutrition solutions formulated tocontain no amino acids or to contain no large neutral amino acids,protein-free liquids (e.g., water, soda, coffee), and oral dietscontaining only carbohydrates and fats. In a preferred embodiment, thepatient is caused to fast for 15 to 20 hours after the administration ofstep (a) and the fasting is followed by administration of protein-freediet (e.g., amino acid-free total parenteral nutrition solutions orother alternatives recited hereinbefore) for 4 to 8 hours with thelatter providing sustainment for the subsequent melphalan administrationand to mitigate sickness being caused by the fasting, and verypreferably, the protein-free diet is continued for up to 4 hours aftermelphalan administration so the plasma large neutral amino acids are notreplenished prior to the uptake of the melphalan.

The invention is illustrated in the following examples:

EXAMPLE I

The kinetic constants K_(m), V_(max), and (V_(max))/(Km) for L-aminoacid oxidase with various amino acid substrates were determined, whereK_(m) stands for the concentration in mM of substrate required forreaction at a rate of 0.5 V_(max) and V_(max) is the maximum rate ofproduct formation achieved per mg of L-amino oxidase as theconcentration of substrate approaches infinity, and (V_(max))/(K_(m)) isthe best measure of L-amino acid oxidase activity toward a substrate.K_(m) and V_(max) were determined by plotting 1/rate vs. 1/[substrate]to give a line which intersects the Y-axis at 1/V_(max) and intersectsthe X-axis at -1/K_(m). The reaction mixtures utilized contained in afinal volume of 1.0 ml, the following: 100 mM glycylglycine buffer, pH7.5, 0.3 mM NADH, 2 mM α-ketoglutarate, 0.5 mM ADP, 20 I.U. glutamatedehydrogenase (Sigma Chemicals, St. Louis, Mo.), and L-amino acidsubstrate or melphalan at concentrations ranging from 0.1 to 16 mM. Thetemperature was 37° C. In this system L-amino acid oxidase convertedL-amino acid or melphalan to the corresponding α-keto acid and ammonia;ammonia then reacted stoichiometrically with α-ketoglutarate and NADH ina reaction catalyzed by glutamate dehydrogenase to give NAD⁺ andglutamate. The oxidation of NADH to NAD⁺ is accompanied by a decrease inOD₃₄₀, which was monitored and used to determine the amount of ammoniamade available for reaction (1 mM NADH converted to NAD⁺ causes a ΔOD₃₄₀of 6.2 in the 1 cm cuvettes used).

The data is set forth in Table 1 below:

                  TABLE 1                                                         ______________________________________                                                   K.sub.m  V.sub.max                                                 Substrate  (mM)     (μmol/min · mg)                                                                (V.sub.max)/(K.sub.m)                        ______________________________________                                        L-Alanine  N/A       0           0                                            L-Leucine  0.6      100          167                                          L-Isoleucine                                                                             2.05     33           16                                           L-Valine   19       13           0.7                                          L-Methionine                                                                             0.47     70           150                                          L-Phenylalanine                                                                          0.13     33           261                                          L-Tryptophan                                                                             0.22     43           194                                          L-Tyrosine 0.13     37           295                                          Melphalan  0.30     11           37                                           ______________________________________                                    

The data provides a screening test indicating that L-amino acid oxidasehas potential in vivo for reducing the level of the large neutral aminoacids L-leucine, L-methionine, L-phenylalanine, L-tryptophan andL-tyrosine without unduly degrading melphalan or other amino acids.

EXAMPLE II

Mice (20-25 gm) were administered either 300 μg or 200 μg of pureL-amino acid oxidase by intravenous injection. At 12, 18, 24 and 36hours after administration, the L-amino acid oxidase activity of theplasma was determined.

L-amino acid oxidase activity was determined as follows: Blood wasobtained from the heart in a heparinized syringe. After centrifugation(10,000×g) for 30 seconds, plasma was obtained as the supernatant. Thenassay reaction mixtures were made up (100 μl final volume) containing400 mM Tris HCl buffer, pH 7.5, 1 mM L-[¹⁴ C]leucine (0.08 μCi) and 40μl plasma. The reaction mixtures were incubated for 10 min. at 37° C.The reaction was stopped by addition of 100 μl of 20% trichloroaceticacid and the vials were placed on ice for five min. The reactionmixtures were then centrifuged for 1 min. to sediment precipitatedprotein, and 180 μl of supernatant was loaded onto a small column(0.5×2.5 cm) of Dowex 50W X 8 (200-400 mesh) cation exchange resin (H⁺-form). The columns were washed with 3.8 ml of water, and the effluentwhich contains α-keto-[¹⁴ C]isocaproic acid, the product of L-amino acidoxidase activity on leucine, was collected in a test tube and mixed.Radioactivity in two ml of the mixed effluent was determined by liquidscintillation counting. The amount of product formed (nmol/min) isdetermined from the known specific activity of the L-[¹⁴ C]leucine used.The amount of L-amino acid oxidase is calculated by knowing that 1 μg ofenzyme forms 100 nmol product/min under these conditions.

Since mice of the size used have about 2 ml of blood and 1.2 ml ofplasma, the peak plasma concentration of L-amino acid oxidase iscalculated to be about 250 and 167 μg/ml in the mice given 300 and 200μg of L-amino acid oxidase, respectively. These peak concentrationswould occur immediately following injection.

The results as determined at 12, 18, 24 and 36 hours are shown in FIG. 1wherein the open circles denote results on administration of 200 μg ofL-amino acid oxidase and the filled in squares denote results onadministration of 300 μg of L-amino acid oxidase. The graphs are definedby points representing the mean ±S.D. for data for 3 mice. As indicatedin FIG. 1, L-amino acid oxidase activity on plasma decreases with timeafter 12 hr. but persists for at least 24 hours. By 36 hr., activity wasessentially zero.

Plasma large neutral amino acids were determined at the 12, 18 and 24hours after L-amino acid oxidase administration.

Results for 300 μg intraperitoneal administration of L-amino acidoxidase are set forth in Table 2 below wherein "control" is amino acidlevel in mice where L-amino acid oxidase was not administered.Concentrations are given as μM.

                  TABLE 2                                                         ______________________________________                                        Amino Acid Control   6 hr    24 hr 36 hr 48 hr                                ______________________________________                                        Leucine    222       121     168   234   180                                  Methionine  97        56      67   106   106                                  Phenylalanine                                                                             95        62      46    78    70                                  Tryptophan  70        54      48    62    92                                  Tyrosine   133        71      42    63    87                                  ______________________________________                                    

The above data suggests that plasma large neutral amino acids can beefficiently depleted at the injected dose between 6 and 24 hradministration. In the period from 24 to 36 hours, the remainingactivity is adequate to maintain plasma levels of some large neutralamino acids at a low level. The dose used is not so high that melphalanitself would be degraded rapidly.

EXAMPLE III

Mice (20-30 gm) were administered either 100 μg, 200 μg, 300 μg or 400μg of L-amino acid oxidase by intravenous injection.

Plasma L-amino oxidase activity was determined at intervals thereafteras shown in FIG. 2 wherein the filled in circles denote results onadministration of 100 μg, the filled in squares denote results onadministration of 200 μg, the filled in diamonds denote results onadministration of 300 μg, and the filled in triangles denote results onadministration of 400 μg.

The graphs are defined by points representing the mean ±S.D. for 3 mice.

As shown in FIG. 2, the plasma L-amino oxidase concentration was greaterthan 50 μg/ml at all times up to 9 hours.

The large neutral amino acid levels corresponding to the L-amino oxidaselevels of FIG. 2 are given in Table 3 below:

                                      TABLE 3                                     __________________________________________________________________________             Plasma Amino Acids                                                   Time                                                                              Dose Leucine                                                                              Isoleucine                                                                           Methionine                                                                           Phenylalanine                                                                         Tyrosine                                (hr)                                                                              (units)                                                                            (μM)                                                                              (μm)                                                                              (μM)                                                                              (μM) (μM)                                 __________________________________________________________________________    0   --   244 ± 73                                                                          150 ± 42                                                                          125 ± 39                                                                          144 ± 40                                                                           165 ± 64                             0.5 2    42 ± 6                                                                            103 ± 7                                                                           28 ± 3                                                                            10 ± 0                                                                             5 ± 0                                1   2    75 ± 22                                                                           110 ± 11                                                                          42 ± 16                                                                           13 ± 9                                                                             7 ± 3                                2   2    26 ± 6                                                                            86 ± 3                                                                            22 ± 4                                                                            5 ± 0                                                                              5 ± 0                                3   2    101 ± 62                                                                          125 ± 26                                                                          38 ± 20                                                                           22 ± 25                                                                            13 ± 13                              4   2    148 ± 21                                                                          144 ± 9                                                                           65 ± 17                                                                           40 ± 11                                                                            27 ±  6                              5   2    51 ± 26                                                                           100 ± 26                                                                          22 ± 5                                                                            8 ± 3                                                                              5 ± 0                                24  2    253 ± 16                                                                          165 ± 4                                                                           108 ± 12                                                                          112 ± 25                                                                           90 ± 33                              2   4    13 ± 11                                                                           86 ± 18                                                                           28 ± 14                                                                           5 ± 0                                                                              5 ± 0                                4   4    23 ± 9                                                                            98 ± 12                                                                           18 ± 1                                                                            5 ± 0                                                                              5 ± 0                                12  4    152 ± 52                                                                          90 ± 30                                                                           76 ± 18                                                                           55 ± 16                                                                            64 ± 41                              18  4    165 ± 13                                                                          96 ± 10                                                                           80 ± 11                                                                           60 ± 3                                                                             46 ± 2                               24  4    159 ± 41                                                                          93 ± 22                                                                           66 ± 19                                                                           65 ± 13                                                                            53 ± 11                              36  4    151 ± 11                                                                          83 ± 10                                                                           49 ± 1                                                                            63 ± 2                                                                             55 ±  3                              2   6    5 ± 0                                                                             45 ± 6                                                                            18 ± 1                                                                            5 ± 0                                                                              5 ± 0                                4   6    5 ± 0                                                                             51 ± 14                                                                           16 ± 3                                                                            5 ± 0                                                                              5 ± 0                                12  6    153 ± 32                                                                          95 ± 27                                                                           72 ± 14                                                                           55 ± 6                                                                             48 ± 8                               18  6    157 ± 14                                                                          97 ± 6                                                                            81 ± 13                                                                           51 ± 5                                                                             37 ± 8                               24  6    101 ± 26                                                                          61 ± 13                                                                           54 ± 13                                                                           46 ± 12                                                                            40 ± 5                               36  6    164 ± 44                                                                          88 ± 20                                                                           58 ± 20                                                                           65 ± 7                                                                             78 ± 40                              2   8    5 ± 0                                                                             42 ± 8                                                                            13 ± 5                                                                            5 ± 0                                                                              5 ± 0                                4   8    5 ± 0                                                                             56 ± 17                                                                           14 ± 3                                                                            5 ± 0                                                                              5 ± 0                                __________________________________________________________________________

As indicated in Table 3, the concentration of large neutral amino acidsduring 30 minutes to 5 hours after administration is reduced to a verylow level thereby providing high melphalan transfer constants for plasmato tumor tissue transfer. While melphalan administered in the first 6hours following L-amino acid oxidase administration will be partiallydegraded by the high levels of L-amino acid oxidase present, in somecases the L-amino acid oxidase mediated decrease in plasma amino acidscan cause an increase in tumor uptake of melphalan that more thancompensates for the L-amino acid oxidase mediated partial degradation ofmelphalan.

EXAMPLE IV

Melphalan was allowed to react with L-amino acid oxidase in a reactionmixture (final volume, 100 μl ) containing phosphate buffered saline, pH7.4, 50 μM [¹⁴ C]melphalan and 125 μg/ml (12.5 units/ml) L-amino acidoxidase. At 5, 15 and 30 minutes after formation of the reactionmixture, 10 μl aliquots were removed and fractionated by HPLC.

The HPLC was carried out on a Brownlee C18 reverse phase column underthe following conditions: Solvent, 0.1M ammonium acetate, pH 4.1, with79% methanol; flow rate 1 ml/min for 15 min, then 1.5 ml/min; fractionsof 1 min collected and radioactivity determined by liquid scintillationcounting.

The HPLC eluant was analyzed for radioactivity and peaks associated withmelphalan (elution time, 8-9 min), or its reaction products (products Iand II eluting at 10-12 and 23.5-25 min, respectively) were determined.The control incubation contained no L-amino acid oxidase, but wasanalyzed similarly. Product I is believed to be ketomelphalan, i.e.,4-[bis(2-chloroethyl)amino]phenylacetate.

The results are shown in FIG. 3 wherein LOX stands for L-amino acidoxidase.

As shown in FIG. 3., melphalan showed good stability for at least 30minutes in the absence of L-amino oxidase and in the presence of L-aminoacid oxidase at 12.5 units/ml was degraded at an initial rate of 50%reaction in 5 minutes.

In this concentration range, the rate of L-amino acid oxidase mediatedmelphalan degradation is expected to be essentially directlyproportional to the L-amino acid oxidase concentration. Thus, at aplasma L-amino acid oxidase concentration of 1.25 units/ml, melphalan isexpected to be degraded with a period required for 50% reaction of 50minutes, and at a plasma concentration of 0.25 units/ml, melphalan isexpected to be degraded with a period required for 50% reaction of 250minutes. As shown in FIG. 1, L-amino acid oxidase concentrations are1.25 units/ml (12.5 μg/ml) or less 12 to 15 hours following 200 to 300μg L-amino acid oxidase administration. In particular, as indicated fromFIG. 1, melphalan is expected to be degraded with a period required for50% reaction ranging from 50 to 250 minutes when administered 12 toabout 30 hours after L-amino acid oxidase administration at theconcentrations of L-amino acid oxidase administered in Example II.Studies in man have indicated that the disappearance of most melphalanfrom plasma is 50% complete after 7.7 minutes. This uptake would beexpected to be increased with the plasma large neutral amino acidreduction provided by L-amino acid oxidase. Thus uptake of melphalaninto brain tumors would be more than 50% complete, 75% complete and87.5% complete 7.7 min, 15.4 min and 23.1 min following injection, i.e.within the period indicated for 50% melphalan reaction.

The above provides a rational basis for estimating the appropriate timefollowing L-amino acid oxidase administration, to administer melphalan.Each tumor type will have its characteristic affinity for melphalan.Thus for a tumor type where uptake is relatively fast (50% uptake in 30minutes or less), uptake will be greater than 87% complete in 90minutes. At a plasma concentration of 5 μg/ml (melphalan administrationabout 18 hrs after 167 μg/ml L-amino oxidase injection or about 30 hrsafter 250 μg/ml injection), less than 25% of the melphalan would bemetabolized by the L-amino acid oxidase in 90 minutes (time required for50% reaction of 250 min, k=0.00276 min) in the absence of melphalanuptake. Considering that melphalan was taken up into tumorssimultaneously with L-amino acid oxidase mediated metabolism, the lossof melphalan is expected to be less than 10%. Since L-amino acid oxidasemediated removal of plasma large neutral amino acids will improvemelphalan uptake by more than 10%, the small loss of melphalan due toL-amino acid oxidase mediated metabolism can be considered insignificantprovided the L-amino acid oxidase dose and timing of the melphalaninjection are properly adjusted.

EXAMPLE V

Mice (20-25 gm) were given melphalan and/or L-amino acid oxidase byintraperitoneal injection. Melphalan was administered at 1.0 or 0.5times the previously established LD₁₀ dose (dose where 10% die), namely11.8 and 23.7 μmole/kg body weight, respectively. Highly purifiedL-amino oxidase was given at a dose of 100 μg or 400 μg per mouse in twoseparate experiments. Results for the first experiment are shown in thefirst five entries in Table 4 below. "LOX" means L-amino acid oxidase:

                  TABLE 4                                                         ______________________________________                                                     Mean Nadir Mean Nadir                                                         weight     Percentage                                            Treatment    loss (g)   Weight Loss (%)                                                                            Deaths                                   ______________________________________                                        1.0 LD.sub.10 Melphalan                                                                    9.2        31.8         0/6                                      0.5 LD.sub.10 Melphalan                                                                    4.2        16.6         0/6                                      100 μg LOX                                                                              0.3         1.1         0/6                                      100 μg LOX & 1.0                                                                        5.8        19.8         0/6                                      LD.sub.10 Melphalan                                                           100 μg LOX & 0.5                                                                        4.7        16.3         0/6                                      LD.sub.10 Melphalan                                                           1.0 LD.sub.10 Melphalan                                                                    4.3        14.6         0/6                                      400 μg LOX                                                                              0.0         0.0         0/6                                      400 μg LOX & 1.0                                                                        3.0        12.5         2/6                                      LD.sub.10 Melphalan                                                           ______________________________________                                    

As shown in Table 4, melphalan alone at either dose caused significantweight loss but no deaths in 6 mice; L-amino acid oxidase alone ateither dose caused no deaths and no significant weight loss in the mice;at the lower dose of L-amino acid oxidase, melphalan plus L-amino acidoxidase caused significant weight loss but no deaths in 6 mice; at thehighest doses, melphalan plus L-amino acid oxidase resulted in the deathof 2 of 6 mice but weight loss was not greater than with melphalanalone. Overall, the results indicate that L-amino acid oxidase does notsignificantly increase the toxicity of melphalan at 1.0 or 0.5 times itsLD₁₀ dose. The difference between the results for "1.0 LD₁₀ Melphalan"in the two experiments in Table 4 may be explained by the biologicalvariability which is always seen in these types of experiments.

EXAMPLE VI

Subcutaneous D-54 MG tumors (a human glioma-derived continuous cellline), grown in athymic BALB/C mice were excised and mechanicallyhomogenized in zinc option medium as described in Bullard, D. E., J.Neuropath. Exp. Neurol. 40:410-427, 1981. The homogenate was mixed withan equal amount of 1% methylcellulose and 10 μl of homogenate (about 10⁵tumor cells) injected into the right frontal hemisphere of athymic mice(20-25 gm) for brain, i.e., intracranial (IC) tumors and 50 μl ofhomogenate was injected subcutaneously into the right flank forsubcutaneous (SC) tumors.

The effects on tumor growth and animal survival of melphalan and L-aminoacid oxidase, alone and in combination were determined in groups of micepreviously injected intracranially or subcutaneously with tumor cellline as described above.

L-amino acid oxidase was administered at a dose of 100 μg (83 μg/ml inplasma) or a dose of 400 μg (333 μg/ml in plasma) by intravenousinjection 8 days after injection of tumor cells. Melphalan when usedalone was administered at a dose of 0.5 times or 1.0 times its LD₁₀ doseby intraperitoneal injection 8 days after injection of tumor cells.Melphalan when used in combination with L-amino acid oxidase wasadministered intraperitoneally 2 hours after L-amino acid oxidaseinjection.

The results are shown in Table 5 below wherein LOX means "L-amino acidoxidase", MDTD means "mean days to death", T-C means growth delay to 5times pretreatment volume for treated tumors minus growth delay to 5times pretreatment volume for untreated tumors and regressions meanspercentage of mice where tumors became smaller.

                  TABLE 5                                                         ______________________________________                                                   Intracranial Subcutaneous                                                     D54 MG       D54 MG                                                                      Long Term      Regres-                                  Treatment    MDTD     Survivors T-C  sions (%)                                ______________________________________                                        No Treatment 19.5     0/10      0    0/10  (0%)                               LOX (100 μg i.v.)                                                                       22.0     0/10      0.2  0/10  (0%)                               Melphalan (0.5 LD.sub.10)                                                                  26.5     0/10      7.2  2/10 (20%)                               Melphalan (0.5 LD.sub.10)                                                                  27.0     0/10      8.9  5/10 (50%)                               & LOX (100 μg i.v.)                                                        Melphalan (1.0 LD.sub.10)                                                                  34.0     0/10      13.3 6/10 (50%)                               Melphalan (1.0 LD.sub.10)                                                                  33.0     0/10      14.6 5/8  (63%)                               & LOX (100 μg i.v.)                                                        ______________________________________                                    

As shown in Table 5, L-amino acid oxidase (LOX) alone had little effecton the growth of either intracranial (IC) or subcutaneous (SC) tumors.Melphalan alone at a dose of 0.5 times the LD₁₀ dose caused a 7.0 dayincrease in median survival time of mice with intracranial tumorscompared to untreated control and a 7.2 day growth delay with tworegressions in 10 mice with subcutaneous tumors. Administration ofL-amino acid oxidase with this dose of melphalan increased mediansurvival time to 7.5 days over untreated control for intracranial tumorsand increased the growth delay to 5 times pretreatment volume to 8.9days, with 5 regressions in 10 mice. At a higher dose of melphalan (1.0times the LD₁₀ dose), the administration of L-amino acid oxidase did notimprove the response of intracranial tumors but did improve the responseof subcutaneous tumors.

Example VII

Intracranial (IC) D-54 MG tumors were grown in Balb/C mice as describedin Example VI. Treatment groups were None (Control), Melphalan alone,Melphalan+diet, Melphalan+LOX, and Melphalan+diet+LOX ("LOX" is used tomean L-amino acid oxidase). L-amino acid oxidase was administeredintravenously at a dose of 300 μg per mouse. Melphalan was administeredintraperitoneally at a dose of 1.0 times its LD₁₀ (the dose previouslydetermined to kill 10% of the animals when used alone). Animals werecarefully monitored to determine "days to death." Scheduling for thetreatment groups was as follows: Mice in the "None (Control)" groupreceived no treatment and the "days to death", therefore representsurvival in days after implantation of the intercranial tumors. Animalsin the "Melphalan alone" group received melphalan 6 days after tumorimplantation and the "days to death" values indicate days of survivalafter tumor implantation. Animals in the "Melphalan+diet" group werefasted for 18 hours beginning 5 days after tumor implantation and werethen allowed free access to a protein-free diet (Bio-Serv Inc. catalogue#F2247) for 6 hours after the fast; at that time melphalan wasadministered, and the animals were allowed a further 2 hours access tothe protein-free diet. Animals were then returned to cages withconventional diet and were monitored for survival; "days to death"indicates survival in days following tumor implantation. Animals in the"Melphalan+LOX" group received 300 μg of L-amino acid oxidase 5 daysafter tumor implantation and 24 hours after L-amino acid oxidaseadministration were given melphalan. "Days to death" indicates survivalin days after tumor implantation. Animals in the "Melphalan+diet+LOX"group received 300 μg of L-amino acid oxidase 5 days after tumorimplantation and were fasted for 18 hours after L-amino acid oxidaseadministration. Animals were then allowed access to a protein-free diet(Bio-Serv Inc. Catalogue #F2247) for 6 hours and were injected withmelphalan 24 hours after L-amino acid oxidase administration. Access tothe protein-free diet was continued for 2 hours after melphalanadministration, and the animals were then returned to cages withconventional diet (standard rodent chow) and were monitored forsurvival. Results are set forth in Table 6 below wherein "Days to Death"indicates survival in days after tumor implantation.

                  TABLE 6                                                         ______________________________________                                                               Median   Number of Mice                                                       Days     Surviving Beyond                              Treatment  Days to Death                                                                             to Death Control Range                                 ______________________________________                                        None (Control)                                                                           19,19,21,23,24,24                                                                         24                                                                25,27,29,29                                                        Melphalan alone                                                                          20,20,27,27,36,36                                                                         36       6                                                        41,41,42,42                                                        Melphalan + diet                                                                         17,20,23,26,33,36                                                                         34       6                                                        37,42,46,47                                                        Melphalan +                                                                              19,21,22,30,30,30                                                                         30       7                                             LOX        30,33,36,38                                                        Melphalan +                                                                              19,19,19,36,36,44                                                                         40       7                                             diet + LOX 44,44,46,47                                                        ______________________________________                                    

The results shown in Table 6 indicate that tumor bearing animalsreceiving no treatment died between 19 and 29 days following tumorimplantation with a mean survival of 24 days. Melphalan alone increasedsurvival in some but not all animals; mean survival increased to 36days. Six of the 10 treated animals had survival times greater than anyof the animals receiving no treatment. Some but not all animalsreceiving melphalan after fasting and exposure to a protein-free dietshowed increased survival relative to animals receiving no treatment;mean survival was 34 days. Six of the 10 animals in this group hadsurvival times greater than any of the animals receiving no treatment.Two of the animals had survival times exceeding those observed in thegroup of animals receiving melphalan alone. Some but not all animalsreceiving melphalan+L-amino acid oxidase exhibited survival timesgreater than the range of survival times observed in the animalsreceiving no treatment; mean survival was 30 days. Seven of 10 animalsin this group had survival times greater than any of the animalsreceiving no treatment. Some but not all animals in the group receivingL-amino acid oxidase and given melphalan after a fast and protein-freediet exhibited survival times greater than those observed in the groupreceiving no treatment. Mean survival time was 40 days; a value greaterthan that observed with any other treatment group. Seven of the 10animals exhibited survival times greater than any of the animalsreceiving no treatment. Five of the 10 animals exhibited survival timesgreater than any observed in the group receiving melphalan alone. Mediansurvival time in the group receiving melphalan+dietarymanipulation+L-amino acid oxidase was greater than median survival timein the group receiving melphalan+dietary manipulation alone.

Enzymes which may be substituted for L-amino acid oxidase in the methodherein include amino acid decarboxylases directed at amino acidstransported by the large neutral amino acid carrier, and phenylalanineammonia lyase.

Drugs besides L-melphalan whose transport may be improved on use insteadof melphalan in the method herein include sarcolysin (D,L-melphalan);melphalan (D-melphalan); meta-sarcolysin, which is3-[m-(bis-(2'-chloroethyl)amino)]phenyl-D,L-alanine; andaminochlorambucil, which is a higher homolog of melphalan; and ingeneral nitrogen mustards of any of the large neutral amino acidsmentioned herein, or any anticancer drug transported by the largeneutral amino acid carrier which has a specificity for large neutralalpha-amino acids.

Many variations of inventive embodiments will be obvious to thoseskilled in the art. Thus, the inventive embodiments are defined by theclaims.

What is claimed is:
 1. A method of treating a patient for tumorssusceptible to melphalan, comprising:(a) administering L-amino acidoxidase to said patient at a dosage ranging from about 1 to about 100units/ml of plasma, which is non-toxic and which is sufficient to reducethe plasma level of large neutral amino acids from a normal level to amelphalan transport improving level; (b) administering an anti-tumoreffective amount of melphalan to said patient within about 2 to 36 hoursafter administering L-amino acid oxidase, wherein the plasma level oflarge neural amino acids has been reduced.
 2. The method of claim 1,wherein the dosage of L-amino acid oxidase administered in step (a)ranges from about 10 to about 50 units/ml of plasma.
 3. The method ofclaim 1 wherein the administration of step (b) is carried out from 12 to30 hours after the administration of step (a).
 4. The method of claim 1further comprising, between the administering of step (a) and theadministering of step (b), preventing replenishment of large neutralamino acids to plasma.
 5. The method of claim 4 wherein preventingreplenishment comprises causing the patient to fast.
 6. The method ofclaim 4 wherein preventing replenishment comprises administering to saidpatient a protein-free diet.
 7. The method of claim 5 wherein preventingreplenishment comprises causing the patient to fast followed byadministering a protein-free diet to the patient.
 8. The method of claim1 wherein the tumors are gliomas.